Human African trypanosomiasis, also known as sleeping sickness, is a zoonotic disease affecting thousands of people across sub-Saharan Africa and periodic epidemics have caused significant social and economic disruption. The trypanosome responsible for sleeping sickness is morphologically indistinguishable from closely related parasites found in animals, leading to confusion regarding the origins of human disease outbreaks. This project seeks to improve the tools available for understanding the epidemiology of this neglected disease. The immediate aims are to develop a robust and broadly applicable panel of genetic markers and to validate these in a pilot study in Uganda, a disease-endemic country currently experiencing a severe epidemic. This project will employ next-generation sequencing of multiple trypanosome genomes, selected to encompass the complicated evolutionary relationships observed among trypanosomes in preliminary studies. The genetic polymorphisms identified from genome comparisons will be evaluated for their ability to resolve fine scale population structure and to distinguish between human-infective and animal-restricted parasites in the Ugandan disease focus. In the longer term, the markers developed in this project will provide a foundation for cataloguing the full diversity of trypanosomes in nature, for identifying the pool of trypanosomes that are most relevant for human disease, and for characterizing the relationships among trypanosome populations distributed across eastern Africa. In addition, comparisons among the genomes of animal-restricted and human-disease causing trypanosomes will provide insight into the functional genetic differences conferring human infectivity.